Résumé
Coffee seeds store pure mannan as the principal reserve for seed germination. However, during fruit development, seeds accumulate first galactomannan, which is degalactosylated towards the end of seed development. In this work, a novel alpha-galactosidase from developing coffee fruits has been purified and characterized. Its activity (optimim pH 6-7) peaks at the stage when galactomannan degalactosylation occurs, i.e. between 150 and 170 days after anthesis (DAA). Using an antibody against alpha-galactosidase from guar seeds, we found that the enzyme is present mainly in the cytosol near the cell walls of endospermic cells. These results, together with the range of pH optima, suggest that the enzyme probably acts on nascent galactomannan when it is at the Golgi. Indeed, some of the activity was found in Golgi fractions prepared from developing seeds,although most of it is soluble. The coffee enzyme showed high specificity towards galactomannan isolated from its own seeds and presented little action on other galactomannans. N-terminal and internal sequences were obtained and the corresponding full-length cDNA sequence named CaGAL2 was isolated from endosperm tissue CaGAL2 expression was highly detected mainly in developing endosperm between 150 and 170 DAA at the time coinciding with the peak of enzyme activity. High expression CaGAL2wexpression was also observed in developing endosperm of C. racemosa. However, CaGAL2 expression was not observed in the perisperm and pericarp as well as in other tissues (leaf, root, flower) from C. arabica. The search in public database with the protein CaGAL2 did not show similarity with other reported alpha-galactosidases, suggesting that this is a novel protein. As this is the enzyme that makes coffee insoluble during maturation because it degalactosylates mannan, it may be speculated that the control of expression of CaGAL2 could be used to develop more productive coffee varieties for soluble coffee production. (Texte intégral)